Composite oncolytic herpes virus vectors

ABSTRACT

Pharmaceutical compositions including a herpes simplex virus derived composite oncolytic vector are provided for the treatment of solid tumors in an individual. The HSV-derived composite oncolytic vector includes an HSV-derived amplicon defective viral genome carrying at least one toxic foreign gene, and an HSV-derived mutant helper virus vector that has a mutation in the vhs-1 gene. An HSV-derived mutant helper virus vector that has a mutation in the vhs-1 gene is also provided. A method for the treatment of an individual having a solid tumor is provided and includes administering an HSV-derived amplicon defective viral genome including at least one toxic foreign gene, and an HSV-derived mutant helper virus vector including a mutation in the vhs-1 gene.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a National Phase Application filed under 35 U.S.C. 371 ofInternational Application No. PCT/IL2002/000345, filed May 2, 2002,claiming the benefit under 35 U.S.C. 119(e) of U.S. ProvisionalApplication No. 60/287,717, filed May 2, 2001, the entire content ofeach of which is hereby incorporated herein by reference in itsentirety.

FIELD OF TH INVENTION

The present invention concerns Herpes Simplex Virus (HSV) derivedvectors and use thereof in the treatment of malignant diseases.

LIST OF REFERENCES

The following is a list of prior art publications referred to in thepresent specification.

-   Efstathiou S. et al., U.S. Pat. No. 5,928,913, 1999.-   Frenkel N, Singer O and Kwong, A. D, Gene Therapy 1:540-546, 1994.-   Kwong A. D. and Frenkel N. Proc. Natl. Acad. Sci. USA 84:1926-1930,    1987.-   Kwong, A. D. and Frenker N, J. Virol. 62:912-921, 1988.-   Kwong A. D. et al. J. Virol. 62:912-921, 1988.-   Leib D. A., 1997 U.S. Pat. No. 5,698,431.-   Markert, J. et al., Herpes 8:1, 2001.-   Rabkin S. et al., American Society of Gene Therapy, Program No.    2046, 2000.-   Ranov, N. G., Gene Therapy 11:2389-2401, 2000.-   Read, G. S. and Frenkel N. J., Virol. 46:498-512, 1983.-   Romi, H, Singer, O, rapaport, D and Frenkel, N, J. Virol.    73:7001-7007, 1999.-   Spaete, R. R. and Frenkel, N, Cell 30:295-304, 1982.-   Spear, M. A. et al., WO 0077167, 2000.-   Vlazny, D. A. and Frenkel N, Proc. Natl. Acad. Sci, U.S.A.,    72:742-746, 1981.

The acknowledgement herein of the above art should not be construed asan indication that this art is in any way relevant to the patentabilityof the invention as defined in the appended claims.

BACKGROUND OF THE INVENTION

Cells infected with HSV-1 and HSV-2 (the facial and genital strains ofHSV) are typically induced to express suicidal genes destined to destroythe cell prior significant viral replication. To overcome this effectand to secure the cell for viral replication, the virus undertakes animmediate counter attack by expressing the virion host shutoff (vhs)(UL41) gene—a 58 kDa structural component of the HSV-1 virion with apowerful mRNA destabilization/degradation activity. The vhs protein isshed into the cellular cytoplasm upon viral uncoating during viral entryinto the cells (Read and Frenkel, 1983; Kwong et al. 1988; Kwong andFrenkel, 1987). Based on recent experiments, it seems that the vhsprotein counteracts the cells' suicidal functions by immediatedestabilization/degradation of the infected cell mRNAs, including housekeeping genes and stress related suicidal genes induced post viralinfection and may encode anti apoptotic genes. In consequence of themRNA degradative activity host cell protein synthesis is shutoff, thesuicidal proteins are not produced and the cells survive for a certainperiod of time, allowing viral replication before death of the targetcell.

HSV-1 mutants carrying a mutation in the vhs gene have been developed.Whereas wild type HSV-1 infection is accompanied by host mRNAdegradation HSV mutants which are deficient in the virion host shut-off(vhs) function (“vhs1 mutants”) allow continued cell protein synthesis.One such mutant termed UL41NAB was developed which infection into cellswas shown to be attenuated in its ability to replicate and reactivatefrom latency (Leib, D., 1997).

HSV derived amplicons comprising at least one inserted gene undercontrol of a promoter in association with helper HSV have been disclosed(Spaete and Frenkel, 1982, Frenkel et al., 1994, Vlanzy, D. A. et al.,1981). In one example of such systems, the associated helper virus is ofa restricted replication competence in a normal host cell (Efstathiou S.et al., 1999). In another example, the recombinant HSV vectors aremodified to target and infect a selected cell type (Spear, M. A. 2000).

The incidence of brain tumors is estimated to be 5-14.1 per 100,000.Gliomas account for 40-60% of the primary tumors, 75% of which aremalignant. Gliomas are the most common primary tumor arising in thehuman brain. Malignant gliomas account for 30% of primary brain tumorsin adults, and are divided by grade into two categories, anaplasticastrocytoma and glioblastoma. The estimated incidence of malignantglioma in the United States is 14.7 per 100,000, representingapproximately 10,000-15,000 new cases annually. Despite improvedaggressive surgical therapy, radiotherrapy and chemotherapy, malignantgliomas are almost always fatal; the overall 5 year survival rate forglioblastoma, the most malignant glioma, is less than 5.5% and themedian survival is approximately 52 weeks. These figures have remainedvirtually unchanged over the past three decades. Treatment of systemictumors often fails because of development of central nervous systemmetastases. The advanced stage indicates no curability. Most gliomashave poor prognosis without any completely effective treatment

Recently, HSV viral vectors were evaluated for their efficacy and safetyin clinical use in humans. In one study, HSV derived vectors comprisingHSV mutant viruses deficient in the gene encoding the 34.5 protein (amajor determinant of neuropathology) were tested in patients withrelapsed glioma. This mutant is a mutli-mutated conditionallyreplicating HSV vector termed “G207” which has deletions of both 34.5loci and in the ICP6 (ribonucleotide reductase (RR)) which is requiredfor replication in non-dividing cells (Rabkin, S. et al. 2000). The G207mutant is now being tested in a Phase I Clinical Trial for recurrentglioblastoma in which it has been shown to be non toxic and withoutserious adverse events, but its efficacy has not yet been demonstrated.In addition, insertion of antineoplastic genes (specifically cytokinegenes) into the mutated vector has been proposed (markert, J, 2001).

In another study an HSV type 1 thymidine kinase and gancyclovir genetherapy was evaluated as an adjuvant to surgical resection and radiationin adults with previously untreated glioblastoma multiform (Ranov, N.G., 2000). In a phase III trial patients with untreated glioblastomamultiform received either standard surgical and radiotherapy or standardtherapy plus adjuvant gene therapy during surgery. Clinical safety ofthe treatment was determined and was comparable in both groups but therewere no significant clinical differences between gene therapy andcontrol patients.

HSV derived viral oncolytic vectors having high efficacy for treatmentof human tumors yet maintaining their safety are desired.

SUMMARY AND GENERAL DESCRIPTION OF THE INVENTION

In order to provide a safe and highly efficient HSV derived vector forthe treatment of a malignant disease, it is desired to provide in such avector at least a dual viral weaponry which will enhance the chance ofeliminating a target tumor cell yet remain safe in that it is designednot to replicate in non-dividing non malignant cells.

In accordance with the Invention, such a composite vector is provided.The HSV-derived vector of the invention comprises two main components:one component which is a defective viral genome with multiplereiterations of amplicon type repeat units each carrying inducible toxicgenes with cell destructive capabilities (“the amplicon”) and as asecond component an HSV mutant helper virus which is incapable ofreplication in non-dividing cells or at least has a significantly lowreplication capacity in such cells (“the helper virus”). Such acomposite vector in accordance with the invention will be referred toherein at times as the “Composite oncolytic vector”.

Preferably, the helper virus is mutated in the virion host shut-off(vhs-UL41) genes. Most preferably, the helper virus is double mutatedboth in the vhs gene as well as in the ribonucleotide reductase (RR)genes.

The dual viral arms of such a vector substantively enhance the efficacyof the vector while maintaining its safety. The dual components of thevector of the invention which attack the target malignant cell both byeffective expression of cytotoxic foreign genes on the amplicon whichare expressed in many copies in a short period of time as well as by theability of the mutated helper to drive the cell to cell death(apparently by inducing apoptosis), substantively enhance the efficacyof the vector while maintaining its safety. The term “enhanced efficacy”should be understood to mean an efficacy which is higher than theefficacy of only one component of the vector (i.e. the ampliconcomprising the toxic gene or helper vector).

Such a composite oncolytic vector comprising a combination of ampliconsand mutants of HSV have not been described.

In accordance with one aspect of the invention, a pharmaceuticalcomposition for use in the treatment of a solid tumor in an individualcomprising an effective amount of an HSV derived amplicon defectiveviral genome carrying at least one toxic foreign gene and an HSV-derivedmutant helper virus and a pharmaceutically acceptable carrier, excipientor diluent.

The term “effective amount” relates to an amount of each of the HSVderived viral components which will, upon administration to theindividual, achieve the desired therapeutic effect. With regards to theamplicon, an effective amount will be such which results in a desiredamount of expression of the foreign toxic gene in a short period oftime. The effective amount of the helper component will be such thatenhances the effect of the amplicon (by providing the necessaryfunctions for gene expression) and preferably an amount which rendersthe helper virus cytotoxic to the cell. Wherein the helper virus is thevhs mutant, the effective amount will be such which leads to death ofthe target cell.

In the amplicon component of the composite vector, the defective genomesare engineered to carry foreign toxic genes. The term “foreign toxicgenes” relates to genes which are not naturally expressed by the targetcells and are designed to destroy the cells in a controlled fashion. Anysuch toxin gene may be used in accordance with the invention, and thegene may be chosen by a person versed in the art on the basis of thekind of tumor to be treated as well as additional factors. An example ofsuch toxic gene in an amplicon is the gene encoding the thymidine kinase(TK) which when expressed in the cells renders them sensitive toganciclovir, producing complete inhibition of host DNA replication andthe destruction of the dividing cells. Other types of toxic foreign geneto be placed in the amplicon are, for example, tumor necrosis factor(TNF), TNF related apoptosis inducing ligand (TRAIL), and P53. Suchtoxic genes can be put under the control of the Tet On system, allowingthe expression of the toxic genes only when treated with tetracycline.Other toxin genes may be constructed to be expressed under control ofother suitable promoters or inducers. The amplicon in accordance withthe invention may also comprise a number of toxic genes under thecontrol of one or more promoters. Such toxic genes may also beconstructed under control of cell or tissue specific promoters which areexpressed only in the desired cell or tissue (e.g. a promoter whichcontrols expression of a prostate specific antigen (PSA) only inprostate cells).

The invention also provides use of an HSV derived amplicon defectiveviral genome carrying at least one toxic foreign gene and an HSV-derivedmutant helper virus for the preparation of a pharmaceutical compositionfor the treatment of a solid tumor in an individual.

In accordance with another of its aspects, the present inventionprovides a method for the treatment of an individual having a solidorgan tumor comprising administration of an HSV derived viral vectorcomprising an effective amount of a combination of an HSV derivedamplicon defective viral genome carrying at least one toxic foreign geneand an HSV-derived mutant helper virus. Preferably, the helper viruscomprises a mutation in the vhs gene. Most preferably, the mutant helpervirus carries also a mutation in the RR genes.

The term “treatment” in accordance with the invention should beunderstood to mean any alleviation of a condition of a patient sufferingfrom a solid tumor. Such alleviation may be a reduction in the size ofthe tumor, reduction in the rate of growth of the tumor, alleviation oftumor-related symptoms, prevention of metastasis, etc.

By a preferred embodiment, the helper virus in accordance with theinvention is constructed to carry a mutated virion host shutoff (vhs)gene such a vector is at times referred to as “vhs mutant”. Thecytotoxic effect of the vhs mutant virus was shown to be associated toinduction of pronounced cell apoptosis in the infected cells. Inaddition, due to the mutated vhs gene, the transcribed mRNA of the toxicgene carried by the amplicon component of the composite vector is notimmediately disintegrated and inactivated (as in the case of thenon-mutated vhs gene), thus enabling expression of the toxic gene andenhancement of the apoptopic effect of the vhs component, resulting inenhanced efficacy of the composite vector as a whole.

The helper virus in accordance with the invention may also comprise amutation in the RR gene. The RR enzyme is essential for viralreplication in resting, non-dividing cells, whereas the virus can usethe cellular RR which is active in growing cells. The RR mutation hasbeen introduced in the small RR subunit (I_(L) 39 gene) of the enzyme.This renders it inactive. The use of RR mutation has the vector saferfor use in gene therapy, by not allowing any replicating virus to spreadto neighboring normal cells. The helper vector may contain the RRmutation alone or together with the mutant vhs. Although, (as shown inFIG. 3 below), growth of the vhs mutant in neuronal cells is limited tomake the vector safer, introduction of the RR mutant into the vhs helpermachinery will make it even safer for potential use in gene therapy.

The two components of the composite vector of the invention may eitherbe obtained by infecting cells with the helper virus and transfectingthe same cells with amplicon plasmids or, by dual transfection of cellswith helper virus DNA and amplicon plasmid followed by repeated serialpropogation of the virus and amplicon mixture to generate a stock ofcells comprising both components which can be then administered to theindividual. Alternatively, the amplicon may first be grown in cells of acell line which comprises non infectious vhs mutated HSV helper viruseslacking the Pac-1 and Pac-2 signals and thus not being able to bepackaged. In this manner, it is possible to prepare large quantities ofthe amplicons ex vivo without infectious helper viruses, resulting in anamplicon packaged in the virions of the vhs mutant virus. Such packagedamplicons are infectious, i.e. they can enter into the cells andintroduce both the foreign cytotoxic genes as well as the vhs-1 mutantgene. Such amplicons may be administered to the individual without ahelper virus, i.e. both components of the vector of the invention willbe present in the packaged amplicons.

The HSV derived, composite oncolytic vector of the invention has a widehost range including epithelial, fibroblastic and neuronal cells andthus is suitable for the treatment of various solid organ tumors suchas, for example, brain malignancies including neuroblastoma andglioblastoma multiform, lung, pancreatic, kidney, colon and stomachcancers.

Typically, in accordance with the invention, the vector will beadministered to the individual by local injection directly into thetumor. However, at times components of the vector may also beadministered by other administration routes including systemically,intraveneously (i.v.), subcutaneously (s.c.), intramuscular (i.m.),intraperitoneal (i.p.) or orally. Such components will be prepared inany of the formulations known in the art suitable for the specific routeof administration chosen by the person versed in the art.

The HSV derived composite vector of the invention will typically havethe following characteristics: (Spaete and Frenkel, 1982, Frenkel et al,1994)

(i) The HSV amplicon is a versatile vector which can targetfibroblastic, epithelial and neuronal cells.

(ii) The system consists of a helper virus and constructed defectivegenomes, which contain multiple reiterations of the amplicon DNAsequences.

(iii) Two cis acting signals are required for amplicon propagation inthe presence of a helper virus: a DNA replication origin and thecleavage packaging signals.

The amplicon can use either the OriS or the OriL replication origins.

(iv) The defective virus genomes replicate by the rolling circlemechanism, which yields “endless” concatemeric DNA molecules, withmultiple head to tail repeats of the amplicon sequences, including thecloned transgene sequences.

(v) The helper virus supplies, in trans, the DNA replication andpackaging machinery including replication enzymes (e.g viral DNApolymerase, helicase, primase, ligase and DNA binding proteins) and thepackaging functions, including the proteins and glycoproteins of the HSVvirion.

(vi) The long replicated DNA concatemers are cleaved during thepackaging process. In HSV the cleavage/packaging signals “pac-1” and“pac-2” are present in the a sequences.

(vii) The cleaved DNA molecules range in size from a single to multiplerepeat units, corresponding in their overall size from the size ofindividual amplicons up to the intact HSV-1 genome (152 kb). Todetermine the details of the cleavage/packaging process we have analyzedby pulse field electrophoresis the viral DNA molecules present in cells,which received different size amplicons. The results of theseexperiments have shown that cleavage/packaging had also involved aheadful constraint with the majority of packaged molecules spanning intheir size approximately genome length DNAs in the range of 136-150 kb.Cleavage take place as the viral genomes are “fed” into the structuralvirion in the process of packaging.

(viii) The precise location of the cleavage and consequent packaging isdetermined by the pac-1 and pac-2 signals which are well conserved inall the herpesviruses (Romi et al., 1999). It has been shown thatcleavage occurs 40-44 bp from the pac-1 signal, and 30-35 bp away fromthe pac-2 signal.

(ix) The pac-1 and pac-2 elements are also required for packaging of thehelper virus. The repeat units of the HSV-1 defective genomes can reach17 kb in their size. Viral amplicons of larger size are randomly deletedin the process of DNA replication, until the repeat unit size reaches 17kb (Kwong and Frenkel, 1985). Defective virus genomes containing repeatsof sizes smaller than 17 kb can be stable propagated in virus stocks formore than 50 sequential passages.

The HSV derived amplicon may also carry a marker gene which enablesdetection of the vector. Such a gene may be any of the known markergenes such as, for example, the green fluorescence protein (GFP) markergenes.

In accordance with the invention, the composite oncolytic HSV derivedvector comprising the two above described components may be administeredto an individual in combination with additional treatments orcomponents. One such component may, for example, be an additional viralvector comprising a gene encoding a peptide which enhances the immuneactivity of the treated individual. An example of such a vector is onecapable of infecting lymphotropic cells such as a herpes virus 6 (HHV-6)or HHV-7 derived amplicon (Romi et al., 1999) containing the“immunogenic” gene. The immunogenic gene may for example be a gene whichencodes for an interleukin such as IL-2, IL4, IL-10 or Interferon and,upon administration, enhances the expression of such peptides in thecells. The “immunogenic” vector may be administered in variouscombinations with the HSV-derived vectors. The additional component maybe administered to the individual by any of the administration routesdescribed above and at various times before, during or afteradministration of the composite oncolytic HSV derived vector.

Thus the present invention further provides a combination of twopharmaceutical compositions including a first pharmaceutical compositioncomprising an effective amount of an HSV derived defective viralamplicon genome carrying at least one toxic foreign gene together withan HSV-derived mutant helper vector and a second pharmaceuticalcomposition comprising an effective amount of an viral derived ampliconcarrying a gene encoding for a peptide capable of enhancing the immunesystem of the treated individual, the combination intended foradministering to the individual for treatment of a solid tumor, in whichtreatment said second composition is administered at time T, said time Tbeing before, during or after administration of said firstpharmaceutical composition.

The above combination may be in the form of a package including saidfirst and said second pharmaceutical compositions.

The invention further provides a method for the treatment of a solidtumor in an individual comprising administering to said individual aneffective amount of a first pharmaceutical composition comprising aneffective amount of an HSV derived defective viral amplicon genomecarrying at least one toxic foreign gene together with an HSV-derivedmutant helper vector and at time T before during or thereafteradministering to said individual a second pharmaceutical compositioncomprising an effective amount of a viral derived amplicon carrying agene encoding for a peptide capable of enhancing the immune system ofthe treated individual.

The invention further provides use of a first pharmaceutical compositioncomprising an effective amount of an HSV derived defective viralamplicon genome carrying at least one toxic foreign gene together withan HSV-derived mutant helper vector for the treatment of a solid tumorin an individual, which treatment includes administering to theindividual said first composition and at a time T before, during orthereafter, administering to said individual a second pharmaceuticalcomposition comprising an effective amount of a viral derived ampliconcarrying a gene encoding for a peptide capable of enhancing the immunesystem of the treated individual.

Yet further, the invention provides a kit comprising a firstpharmaceutical composition comprising an effective amount of an HSVderived defective viral amplicon genome carrying at least one toxicforeign gene together with an HSV-derived mutant helper vector and asecond pharmaceutical composition comprising an effective amount of aviral derived amplicon carrying a gene encoding for a peptide capable ofenhancing the immune system of the treated individual, together withdirections for use.

Furthermore, the additional treatment administered to the treatedindividual may be any other treatment typically administered toindividuals having a solid tumor, such as for example, treatmentsintended to enhance the level of immune response (e.g. Interferon) ortreatment and targeting of the tumor cells such as radiation orchemotherapy.

In the following, the invention will be exemplified with reference tothe following non limiting examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation showing an example of the twocomponents of the composite oncolytic HSV-1 derived vector of theinvention. One component shown is an HSV-1 amplicon carrying multiplereiterations of at least one toxic gene and the second component is amutant HSV-1 derived helper virus with replication capacity in dividingcells and carrying a mutation in the virion host shutoff (vhs) functionwhich induces cellular suicidal death functions as well as a mutation inthe ribonucleotide reductase (RR) gene.

FIG. 2 is a schematic representation showing an example of one componentof the composite oncolytic vector of the invention being an HSV-derivedoncolytic HSV-1 amplicon carrying the p53, a viral origin of replication(ori) and packaging signals (pac).

FIG. 3 is a schematic representation showing the replication of a vhs-1mutant helper virus after infection into mouse cerebral granular neuronsas compared to a wild type HSV (KOS) vector infected into such cells.The replication was measured at different multiplicities of infection(m.o.i.) and at different times after infection.

FIG. 4 is a graphic representation showing the viability of non infectedmouse neuron cells (mock-control) and mouse neuron cells followinginfection with the vhs-1 mutant vector in different multiplicities ofinfection at different times after infection of the cells and asmeasured by the MTT assay.

FIG. 5 is a schematic representation showing the viability of neuroncells after infection of the cells with either a HSV derived vector(KOS) or a vhs-1 mutant vector at various times after infection of thecells and as measured by the trypan blue assay as compared to noninfected (mock) control cells.

FIG. 6 shows photographs of mouse neuronal cell cultures infected withthe KOS or vhs-1 viral vectors at various times after infection and DAPIstaining. Apoptosis is seen in the vhs-1 infected cells.

FIG. 7 is a schematic representation showing the percent of cell deathof H1299 human lung carcinoma cells infected with the vhs-1 mutanthelper virus alone or in combination with a HSV-1 amplicon vectorcarrying the p53 gene. The vectors were infected at a multiplicity ofinfection of 1 plaque forming unit (pfu)/cell (FIG. 7A) and 10 pfu/cell(FIG. 7B) and the number of viable dead cells were determined at varioustimes after infection by the trypan blue assay.

FIG. 8 is a schematic representation showing cell death of H1299 humanlung carcinoma cells after infection with an HSV derived vector carryingthe 34.5 mutation alone or in combination with an HSV-1 derived ampliconcomprising the p53 gene in a multiplicity of infection of 1 pfu/cell(FIG. 8A) or 3 pfu/cell (FIG. 8B) at various times after infection ofthe cells and as determined by the trypan blue assay.

FIG. 9 is a schematic representation showing the viability of H1299human lung carcinoma cells after infection with the doubly mutated HSV-1vector carrying the 34.5 and vhs mutant genes in combination with anHSV-1 amplicon carrying the p53 cytotoxic gene at a multiplicity ofinfection of 1 pfu/cell (FIG. 9A) or 10 pfu/cell (FIG. 9B) at varioustimes after infection of the cells and as determined by the trypan blueassay.

EXAMPLES Example 1 Materials and Methods

Cell cultures:—Primary cultures highly enriched for cerebellar granularneurons were prepared from 8 days old BALB-C mice. Cultures were madefrom mouse brains. The cells were trypsinized and plated on dishescoated with poly-L-lysine in standard medium (basal medium Eagle's, 10%fetal calf serum, 25 mM KCl, 2 mM glutamine, 50 μg/ml gentamycin and 250ng/ml amphotericine B supplemented with 1 mg/ml glucose.Cytosine-β-arabinofuramoside (Ara-C) (10 μM was added to the medium18-22 h after plating to prevent replication of non neuronal cells.Viruses:—HSV-1 (KOS) served as the wild type virus. The virionassociated host shutoff mutant was derived in our laboratory from −1(KOS) by general BudR mutagenesis and selection of mutants which did notshutoff host protein synthesis in the presence of actinomycin D toreassure that this is a virion function brought into the cells withinthe infecting virions (Read and Frenkel, 1983). Virus stocks were madewith limited passaging, employing Vero cells at an input multiplicity ofinfection (m.o.i.) of 0.01 pfu/cells.HSV infection of cerebellar granule cells:—The granule neurons wereinfected four days after plating the neurons. The number of viable cellswas determined each experiment, employing trypan blue exclusion assay.The neurons were washed twice with conditioned medium to remove theAra-C and then exposed to the appropriate virus m.o.i., as stated in thetext. Infection was in 199V medium with 1% fetal serum. The cells wereinfected for two hours at 37° C. The innoculum was then removed andconditioned medium added prior to further incubation at 37° C.Assay of infectious virus yield:—At different times p.i. the infectedcerebellar granule neurons were harvested and disrupted by three cyclesof freezing and thawing, to release the virus. Infectious virus wastitered by plaque assays in Vero cells.Trypan blue viability assay:—Neuron survival was determined by trypanblue exclusion assay. Cells were incubated for 10 min in a solution of0.1% of trypan blue in phosphate buffer saline (PBS), pH-7.4 and thenwashed twice with PBS. Three randomly chosen fields which containedapproximately 500 cells each were analyzed by phase-contrast and brightfield microscopy. Cells excluding the dark blue dye were counted asviable, whereas blue-stained cells were scored as dead.MTT assay:—A modification of previously described procedure was used:neuronal cultures (in 96 well plates) were incubated for 60 min. at 37°C. with 0.5 mg/ml MTT in standard medium. The MTT solution was aspiratedand the cells were lysed in 200 ml DMSO. The amount of MTT formazan wasquantified by determining the absorbency at 490/690 in a Bio-tekmicroplate reader (Wiooski, Vt., USA).DNA staining with DAPI:—Cells were grown on glass cover slip coated withpoly-1-lysin. The cells were infected. Upon completion of theexperiments, the cells were washed with phosphate saline buffer pH-7.4(PBS) and fixed for 10 min in 4% formaldehyde (in PBS). After fixationthe neurons were washed with PBS, stained for 5 min with 10 μg/ml DAPI(4,6-diamino-2-phenylindol), and washed twice with PBS; a drop of eitherN-propyl gallat or glycerol was added to enhance fluorescence, which wasdetected by UV light microscopy. When completed the cells were washedwith PBS and fixed in 45 formaldehyde in PBS. After fixation and washingstained with 10 mg/ml DAPI (washed, and a drop of glycerol was added toenhance the fluorescence which was detected by UV light microscopy.

Results

The Replication of Wild Type (Wt) and Vhs-1 Mutant Viruses in MouseCerebellar Granule Neurons:

The replication of wt HSV-1 (KOS) and the vhs-1 mutant viruses in mousecerebellar granule neurons, was analyzed at different multiplicities ofinfection (m.o.i.)s. FIG. 3 compares infectious virus yield in theneuronal cells up to 48-hrs post infection (p.i.) with input m.o.i. of0.1, 1 and 10 PFU/cell. Titration of the resultant virus stocks was donein Vero cells. The results have shown that the wt virus replicatedproductively in the neuronal cells. Infectious virus yield was highestin the cultures infected with an input m.o.i. of 0.1 pfu/cell (1428 foldamplification of the input virus). They were lower with the inputm.o.i.s of 1 and 10 PFU/cell (62 and 4.6 fold amplification of the inputvirus, respectively, by 48 hrs p.i. In contrast, the vhs-1 mutant didnot replicate well in the neuronal samples, with infectious virus yieldcorresponding to 2 fold-input virus in cells infected with 0.1 PFU/cellvhs-1 mutant virus and no amplification of virus in cells infected with1 and 10 PFU/cell respectively. Based on the data it can be concludedthat the vhs-1 mutant posses only limited capacity to replicate in thebrain cells.

The Induction of Programmed Cell Death (Apoptosis): MTT Assays:

Because the vhs function causes destabilization/degradation of host cellmRNAs it was of interest to determine whether the infected cells wereinduced to undergo into a programmed cell death, and whether the vhs-1mutant was more toxic to the cerebellar granule neurons. Duplicate 96well cultures of the purified cereberal neuron cultures were infectedwith HSV-1 (KOS), or the vhs-1 mutant viruses. Cell viability wasmeasured by MTT formazan incorporation at 12, 24, 36 and 48 hrs p.i.,quantified by 490/690 absorbency in Bio-tek microplate reader. Theexperiment was repeated several times with similar results employingdifferent input m.o.i.s An exemplary experiment involved infection of 96well monolayers of purified granular neurons cultures at input m.o.i. of1, 5 and 10 PFU/cell of HSV-1 (KOS) or the vhs-1 mutant viruses. Asshown in FIG. 4.

Whereas the neuronal infection with the wt HSV-1 (KOS) did not causeapoptosis by 48 hrs p.i., the vhs-1 mutant virus infection wasaccompanied with pronounced (down to 50%) apoptosis.

Trypan Blue Assay:

In parallel to the MIT assay neuronal viability was determined by trypanblue exclusion assay. Cells excluding the dark blue dye were counted asviable, whereas blue-stained cells were scored as dead. As shown in FIG.5, whereas KOS virus infection at m.o.i. of 3 PFU/cell did not causesubstantial cell death by 48 hrs p.i., close to 50 and 70% death ofneuronal cells have died in the vhs-1 infected cultures.

Characterization of Cell Death: DAPI Assays

To examine whether the infection produced apoptotic death, the neuronalis cultures were grown on a cover slip, with poly-L-lysin support.Following infection with wt and vhs mutant viruses for different lengthof time the cells were fixed with formaldehyde, stained with DAPI(4,6-diamino-2-phenylindol) and examined in the fluorescent microscope.FIG. 6 shows that nuclei of uninfected granule neurons appeared uniformin size, with an oval shape, and were rather homogeneously stained withmoderate intensity and spotted with glowing areas, typical of mousecells. Twelve hours after exposure to KOS the cells appeared unchangedwhereas in the cells exposed to vhs viruses, some neurons' nuclei losttheir oval shape and appeared like bright round spots. As the deathprocess progressed, the number of the disintegrated nuclei (roundcompartments) increased. Twenty-four hours after exposure to vhsviruses, all the nuclei of the underwent margination, fragmentation andcondensation into individual particles while some of the KOS exposedcells showed first signs of the deterioration process. These results areindistinguishable from the observations of the DAPI staining of cerebralgranule neurons deprived of high potassium, which leads to apoptoticneuronal death. Taken together, our results show that virusinfection-induced death has apoptotic characteristics.

The effect of viral infections on degradation of mRNA of house keepinggenes as well as stress related genes induced post infection (such astubulin and the heatshock 70 proteins (HSP-70)), in mock infected cellsor in cells infected with HSV-1 (KOS), HSV-2 (6) and the vhs-1 mutantwas analyzed (results not shown). Several conclusions can be drawn fromthis exemplary experiment: (i) the vhs function does not require viralgene expression post-infection inasmuch as mRNA degradation occurs alsowhen the cells were infected in the presence of actinomycin D,preventing altogether the transcription of host viral genespost-infection. (ii) The vhs function degrades genes such as tubulin.(iii) The heatshock “stress” mRNA was induced post-infection 70 proteinwhich is induced in response to viral infection. Actinomycin D treatmentprevented its accumulate in KOS and vhs-1 mutant virus infection.

As shown in the above figures, the above experiments employingcerebellar granule neurons of 8 day old BalbC mice show that: (i) the wtvirus replicates well in the cerebellar granule neurons whereas noreplication of the vhs-1 mutant occurred even by 48 hours post Infection(ii) wt virus infection does not induce apoptosis whereas mutant virusinfection has induced pronounced cell death as measured by mitochondrialMIT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenltetrazolum bromide) assaysof cell viability (iii) Similarly, trypan blue assays revealed no celldeath following wt virus infection, compared to pronounced death at thevhs-1 infection. (iv) The neuronal cell death reflected apoptosisassociated with cell deterioration and nuclei breakage as judged by Dapifluorescence. The vhs-1 mutant virus infection causes more pronouncedapoptosis and earlier that the limited apoptosis caused by the wt virus.

The above results also show that wt virus inhibits host gene expressionpost infection by letting the vhs RNase startdestabilization/degradation of infected cell mRNAs including the hostdeath genes induced in response to viral infection. In contrast, thecellular mRNAs are expressed efficiently in vhs-1 mutant virusinfection, resulting in pronounced apoptosis. As noted above cellsurvival was advantageous for virus replication and whereas wt viruswhich did not cause apoptosis replicated well in the cerebellar neuronsthe vhs-1 mutant, which kills the cell, did not replicate in mouseneurons even by 48 hours post infection.

Example 2 Materials and Methods

H1299 human lung carcinoma cell line cells were infected with HSV-1mutants (exemplified by vhs-1, γ34.5 and the 34.5, vhs double mutant) atmultiplicity of infection of 1 and 10 pfu/cell. In lung cells, thehelper virus must carry a mutation which prevents its replication inorder to be safe. The number of viable and dead cells were determined at14, 24, 36, 48, 72 and 96 hours post infection by typan blue assay.

Trypan Blue Assay

The cells were incubated 1 min. in a solution containing 0.1% of trypanblue in phosphate buffer saline. Then 500 cells were counted by brightfield microscope. Cells excluding the blue dye were counted as viable,whereas blue stained cells were scored as dead.

Results

The results are shown in FIGS. 7-9 and can be summarized as follows:

-   (1) Good expression of the p53 gene (in all the samples containing    the amplicon) in the lung cells is shown in the Western Blot in the    human lung carcinoma cells.-   (2) Concerning cell death in the infections:    -   (i) The vhs-1 infections (FIG. 7) at 1 (FIG. 7A) and 10 (FIG.        7B) pfu/cell resulted in pronounced cell death operating        exponentially, peaking at 3 and 4 days post infection (90, and        93% cell death respectively).    -   (ii) The addition of the p53 containing amplicon increased cell        death: as shown in the figures, the dual infections of the vhs-1        helper and the p53 amplicon resulted at each time point with        increased cell death (e.g. 2 days post infection (p.i.) there        was 20 and 40% cell death in the cultures infected with vhs and        vhs+p53 respectively).    -   (iii) As shown in FIG. 8, infection with the 34.5 at 1 and 10        pfu/cell resulted in incomplete death, which gradually increased        reaching 30% death at the 1 pfu/cell infection, and 60% death at        the 3 pfu/cell infection. The death was slowly plateauing at 3        to 4 days post infection.    -   (iv) Dual infection of the cells with the 34.5 mutant helper and        the p53 amplicon resulted in significantly increased death.        Death increased exponentially between 2 and 3 days post        infection reaching 88% at 3 days p.i. and reaching a plateau at        90% at 4 days p.i.    -   (v) As shown in FIG. 9, the lung carcinoma cells infected with        the double mutant 34.5×vhs underwent exponential cell death        already between 2 and 3 days p.i. Death was 10% by 2 days post        infection and increased exponentially to 90% by 3 days p.i.    -   (vi) Death increased more rapidly and reached additional        efficacies by the addition of the p53 amplicon reaching 93 and        100% 3 and 4 days p.i. respectively.-   (3) Similar results were obtained using an MTT assay (measuring cell    viability by the functionality of mitochondria) (not shown).

Example 3

HSV-1 amplicons containing the thymidine kinase (tk) gene wereconstructed. The amplicon is infected into various cells and tk geneexpression in the in infected cells is evaluated by Western Blotanalysis as described above.

In addition, gancyclovir induced death of the cells infected with the tkcomprising amplicon alone or in combination with an HSV helper viruscontaining a mutated hvs gene is evaluated using the trypan blue or MTTassays as described above.

Example 4

An HSV-1 amplicon containing a gene encoding TNF has been constructed.The expression of the TNF gene in various infected cells as well as celldeath of cells infected with the TNF comprising amplicon alone or incombination with an HSV derived helper vector containing a mutated hvsgene is evaluated as described above.

Example 5 In Vivo Effect of the HSV vhs Mutant Vector on Growth ofTumors in Vivo

Nude mice are injected subcutaneously with cells of a human glioblastomacell line. At various stages of growth of the tumors, the mice aredivided into the following groups:

-   -   (a) control mice receiving mock injections.    -   (b) mice receiving injections of the vhs-1 mutant HSV vector;    -   (c) mice receiving injections of a combination of the HSV        amplicon carrying one or more toxic genes and the vhs-1 mutant        HSV helper virus;    -   (d) mice receiving injections of the pure HSV amplicon devoid of        infectious helper virus but carrying one or more toxic genes and        the vhs-1 mutant protein in the virion encapsulating the        amplicon (grown in cells comprising a pac-1, pac-2 deleted and        vhs mutant helper virus);    -   (e) mice receiving each of the treatments of (a)-(d) together        with a systemic infection of an HVV-6 or HVV-7 derived amplicon        containing an IL-2 encoding gene at various times before,        together with or after the injection of the HSV vectors.

The viral vectors are injected directly into the tumors of the mice andthe development of the tumors in each of the groups is determined atvarious times after injection and compared to the development of thetumors in the control group.

In an additional in vivo experiment, the above-mentioned vectors areinjected into tumors developed from pancreatic malignant cells or longmalignant cells in nude mice, as another example to highly malignanttumors within internal organs.

1. A pharmaceutical composition for the treatment of a solid tumor in anindividual, comprising: an effective amount of an HSV1 amplicondefective viral genome comprising at least one toxic foreign geneselected from the group consisting of thymidine kinase (TK) and/or P53and an HSV mutant helper virus vector comprising the vhs-1 mutationUL41NHB in the virion host shut off gene and an inactivating mutation inthe small ribonucleotide reductase (RR) subunit gene; and apharmaceutically acceptable carrier, excipient or diluent, whereinexpression of the at least one toxic foreign gene inhibits tumor growthupon direct intra-tumor injection of the pharmaceutical composition tothe solid tumor.
 2. The pharmaceutical composition of claim 1, whereinthe solid tumor is a brain tumor.
 3. A method for the treatment of anindividual having a solid tumor, comprising: administeringintra-tumorally by direct injection to the solid tumor in theindividual, an HSV1 viral vector comprising an effective amount of acombination of an HSV1 amplicon defective viral genome comprising atleast one cytotoxic foreign gene selected from the group consisting ofthymidine kinase (TK) and/or P53, and an HSV mutant helper virus vectorcomprising the vhs-1 mutation UL41NHB in the virion host shut off geneand an inactivating mutation in the small ribonucleotide reductase (RR)subunit gene, wherein expression of the at least one toxic foreign geneinhibits tumor growth.
 4. The method according to claim 3, wherein thesolid tumor is a brain tumor.
 5. A combination pharmaceuticalcomposition for the treatment of a solid tumor in an individual,comprising: a first pharmaceutical composition comprising an effectiveamount of an HSV1 defective viral amplicon genome comprising at leastone toxic foreign gene selected from the group consisting of thymidinekinase (TK) and/or P53 and an HSV mutant helper virus vector comprisingthe vhs-1 mutation UL41NHB in the virion host shut off gene and aninactivating mutation in the small ribonucleotide reductase (RR) subunitgene; and a second pharmaceutical composition comprising an effectiveamount of an HHV-6 or HHV-7 amplicon vector comprising a gene encodingfor a peptide capable of enhancing the immune system of a treatedindividual, wherein expression of the at least one toxic foreign geneinhibits tumor growth upon direct intra-tumor injection of the firstpharmaceutical composition to the solid tumor, and wherein said peptidecapable of enhancing the immune system is IL-2, IL-4, IL-10 orinterferon.
 6. The combination pharmaceutical composition of claim 5,further comprising a package comprising the first and the secondpharmaceutical compositions.
 7. A kit, comprising: a firstpharmaceutical composition comprising an effective amount of an HSV1defective viral amplicon genome comprising at least one toxic foreigngene selected from the group consisting of thymidine kinase (TK) and/orP53 and an HSV mutant helper virus vector comprising the vhs-1 mutationUL41NHB in the virion host shut off gene and an inactivating mutation inthe small ribonucleotide reductase (RR) subunit gene; a secondpharmaceutical composition comprising an effective amount of an HHV-6 orHHV-7 amplicon vector comprising a gene encoding for a peptide capableof enhancing the immune system of a treated individual; and directionsfor use of the kit, wherein expression of the at least one toxic foreigngene inhibits tumor growth upon direct intra-tumor injection of thefirst pharmaceutical composition to the solid tumor, and wherein saidpeptide capable of enhancing the immune system is IL-2, IL-4, IL-10 orinterferon.
 8. The method of claim 3, wherein the HSV1 viral vector isadministered to the individual receiving an additional treatment.
 9. Aninjectable composition, comprising: a HSV mutant helper virus vectorcomprising the vhs-1 mutation UL41NHB in the virion host shut off geneand an inactivating mutation in the small ribonucleotide reductase (RR)subunit gene; and a pharmaceutically acceptable carrier, excipient ordiluent.
 10. A method of treating a solid tumor in a subject,comprising: administering the injectable composition according to claim9 to the subject in need thereof, the administering comprisingintra-tumoral administration by direct injection.
 11. The methodaccording to claim 10, wherein the solid tumor is a brain tumor.